The authors thank the Sharon group members for their critical review, and for their contributions to the manuscript. We are grateful for the support of the Morasha and Bikura Programs, the Israel Science Foundation (Grant Nos. 1823/07 and 378/08), the Josef Cohn Minerva Center for Biomembrane Research, the Chais Family Fellows Program for New Scientists, the Abraham and Sonia Rochlin Foundation; the Wolfson Family Charitable Trust; the Helen and Milton A. Kimmelman Center for Biomolecular Structure and Assembly; the estate of Shlomo and Sabine Beirzwinsky; Meil de Botton Aynsley, and Karen Siem, UK.

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No conflicts of interest declared.

The protocol described here enables to define the collision cross section of proteins or protein complexes with an unknown three dimensional structure, with the aim of providing information on their overall shape, subunit packing and topology. To this end once collision cross section values are depicted it is necessary to convert these values to structural details. This process requires additional experimental efforts as well as computational analysis, which are briefly discussed below. 
To begin with, it is recommended to analyze proteins or protein complexes with known structures. These measurements can provide a useful quality control of the methodology and will enable accuracy assessment of the acquisition parameters by comparing theoretical and measured &Omega; values. The theoretical cross sectional areas can be calculated from the crystal structure coordinates using the MOBCAL15,16 software, which is an open source FORTRAN based software allowing code editing according to the operator needs. For running such calculations it is required to modify the program such that the number of iterative calculations performed per input structure is increased and that coordinate files containing large number of atoms are accepted1.
An IM-MS strategy for defining topological arrangements of subunits within multicomponent assemblies has been recently proposed4,6. The method involves the monitoring of dissociation pathways of protein assemblies to smaller components. This dissociation is achieved through controlled adjustment of solution phase conditions, which gives rise to a distribution of subcomplexes reflective of the "building blocks" of the assemblies. The simultaneous measurement of &Omega; values of both the intact complex and disassembly products generates structural restraints which are then used for calculating topological models of the protein complexes. The basic assumption underlying this methodology is that the generated subcomplexes retain their native-like confirmations, and indeed recent studies have demonstrated that the solution structure of the disassembly products is maintained and no major rearrangement in either solution or gas phases have occurred4,6.
The last step in the assignment of quaternary structure to gas-phase protein complex ions is fitting the collision cross section values to computer generated models. Modeling approaches are employed in order to explore the different possible topological arrangements of subunits and their in silico &Omega; values are calculated and compared to the experimental ones. Currently only a few computational approaches are used, like the spheretype coarse-grained method that approximates the diameter of subunits1,8. On the whole, this field is still in its early years and further development is required to make this approach generic, and applicable to a wide range of complexes.

t-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

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T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

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24.3K Views.  Weizmann Institute of Science. Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.